A number of polypeptides from a variety of sources are used to treat disease in humans. If the polypeptide is derived from a heterologous or non-self source, patients readily develop an immune response to the polypeptide. In fact, in many cases, an immune response to the polypeptide is a desired therapeutic outcome. However, there are situations where an immune response to a polypeptide intended for therapeutic use is undesirable and could be very detrimental. For example, polypeptides intended for therapeutic use also include polypeptides that have endogenous or xe2x80x9cselfxe2x80x9d counterparts. It is generally thought that an immune response is not generated to self or endogenous polypeptides, except in the case of autoimmune disease. If an immune response were generated upon administration of such polypeptides, adverse consequences could result including development of an autoimmune response.
Many factors contribute to immune responses to polypeptides. The presence of immunodominant epitopes in a polypeptide is one of the key factors in antibody responses. An immunodominant epitope is an epitope that more frequently elicits and binds to antibodies in a human or animal or population thereof when compared with other epitopes. Immunodominant epitopes in polypeptides are typically identified after administration of the polypeptide to the human or animal.
Immunodominant epitopes have been mapped on proteins using antibodies from treated animals. For example, staphylokinase is a bacterial polypeptide derived from Staphylococcus aureus and is used to treat myocardial infarction. Because staphylokinase is a heterologous or non-self polypeptide when administered to humans, treated patients readily develop an immune response to this polypeptide. This immune response can result in adverse reactions to further therapeutic treatment with the polypeptide. As described in U.S. Pat. No. 5,951,980, the immunodominant epitopes identified using antibodies from treated patients can be modified to reduce immunogenicity of the polypeptide.
In some cases, immunodominant epitopes on heterologous polypeptides have been masked or modified to shift the immune response to other epitopes in the polypeptide. As described in PCT 99/38978, patients exhibit antibody responses to IgG and IgE epitopes on allergens from heterologous sources such as peanuts, latex and food proteins. Once an IgE epitope is identified using the patient""s antibodies, the IgE epitope can be modified or masked to shift the immune response from the IgE epitope to other epitopes on the allergen. Similarly, the immune response to the immunodominant epitopes on pathogens, such as HIV, can be shifted to other epitopes on the pathogens by modifying or masking the immunodominant epitopes as described in U.S. Pat. No. 5,853,724. An immune response to these polypeptides is still a desired outcome.
Immunodominant epitopes have also been identified on some endogenous or self-proteins associated with autoimmune disease. Thyroid peroxidase is an endogenous molecule associated with autoimmune thyroid disease. For a review see McIntosh, R. S. et al, Thyroid 7:471 (1997). Immunodominant regions have been mapped on thyroid peroxidase using antibodies from patients with autoimmune disease. See Hobby, P. et al, Endocrinology 141:2018 (2000) and Nishikawa, et al, Endocrinology 137:1000 (1996). Mapping of these epitopes has been used to study the factors that might contribute to the development of autoimmune disease. See Jaume, J. C. et al, J. Clinical Endocrinology 84:1424 (1999). Factors that make self or endogenous polypeptides immunogenic in autoimmune disease are complex and not well understood.
Immunodominant epitopes on polypeptides intended for therapeutic use have not previously been identified before administration of these polypeptides to patients. In the case of polypeptides that have a sequence identical to all or a portion of a self or endogenous polypeptide, it was thought that these polypeptides would not elicit an immune response upon administration to patients.
One example of a recombinant human polypeptide used therapeutically is human thrombopoietin (TPO). A recombinant TPO has a sequence that is identical to a human thrombopoietin and is produced in CHO cells. TPO is an endogenous hemopoietic growth factor that stimulates proliferation and maturation of megakaryocytes and production of platelets. A recombinant human TPO is made up of 332 amino acids linked in a single polypeptide chain. Approximately in the middle of a molecule, there is a dibasic site of arginine 153 and arginine 154 that divide the molecule into a n-terminal and c-terminal epitopes. The n-terminal epitope is called the EPO epitope because of its similarity to erythropoietin or it is also called truncated TPO. The n-terminal epitope contains receptor-binding sites and represents the biologically active portion of the molecule. The c-terminal epitope is heavily glycosylated.
The major causes of thrombocytopenia in patients are impaired production of platelets by bone marrow, platelets sequestration in the spleen and destruction of platelets by autoimmune responses. Recombinant TPO stimulates production of platelets and is intended for the treatment of thrombocytopenia in patients undergoing chemotherapy. It is well known that repeated cycles of radiation and chemotherapy result in myelosuppression which limits dose intensity of chemotherapeutic agents. Administration of thrombopoietin, especially recombinant thrombopoietin, has resulted in improved tolerance of the patients for chemotherapy as described in WO 98/52598.
However, if administration of recombinant TPO or other therapeutic polypeptides can result in an immune response, patients may become refractory to the treatment or develop other adverse consequences. Thus a need exists to identify immunodominant epitopes in polypeptides intended for therapeutic use before administration to patients and to modify the polypeptides to reduce the immune response to the polypeptide.
One object of the present invention, is to develop methods to screen polypeptides intended for therapeutic use to identify immunodominant epitopes in order to reduce the chance of developing of an immune response to the polypeptide.
Another object of the invention is to design such polypeptides to modify the immunodominant epitopes in order to reduce the immune response to such polypeptides while retaining a substantial therapeutic activity when administered in vivo.
These and other objects will become apparent in the description in the embodiments of the invention provided herein.
The present invention is based on the unexpected and surprising finding that naive humans and animals can have pre-existing antibodies to a polypeptide intended for therapeutic use such as recombinant human polypeptides. It was also surprising that patients develop an antibody response after dosing or administration of these polypeptides because it was generally thought that an immune response would not be formed to a polypeptide having a sequence identical to that of the endogenous or self protein. These findings indicated a need to screen polypeptides intended for therapeutic use to identify immunodominant epitopes before administration to patients. The antibodies from naive patients with pre-existing antibodies to the polypeptide can advantageously be used to identify immunodominant epitopes on polypeptides before they are administered to patients. The identification of immunodominant epitopes prior to clinical application of the polypeptide can be used in designing less immunogenic molecules.
The invention provides for methods of identifying immunodominant epitopes in polypeptides, preferably polypeptides intended for therapeutic use. A method of the invention is a method for identifying at least one immunodominant epitope in a polypeptide by using an antibody or population of antibodies from a naive human or animal or population thereof. Immunodominant epitopes are those epitopes that more frequently bind to or are recognized by antibodies or a population of antibodies than other epitopes in the polypeptide. Immunodominant epitopes are also identified using both an antibody and a population of antibodies from a naive human or animal and an antibody or population thereof from a human or animal dosed with the polypeptide. An immunodominant epitope is selected for modification. The polypeptide is preferably a recombinant polypeptide intended for therapeutic use with a sequence identical to all or a portion of the native sequence of an endogenous polypeptide. The animal is preferably human.
Immunodominant epitopes are also identified by utilizing methods of predicting epitopes in polypeptides through the use of, for example, algorithms. Predicting epitopes in polypeptides reduces the amount of time and resources needed to identify immunodominant epitopes. Accordingly, a method of the invention involves providing a data set of the polypeptide, analyzing the data set with an algorithm to identify at least one predicted epitope in the polypeptide, and determining whether the predicted epitope is an actual immunodominant epitope.
The invention also provides for methods of modifying a polypeptide to reduce an immune response to the polypeptide while retaining a substantial therapeutic activity. A method involves identifying at least one immunodominant epitope in a polypeptide by using an antibody or population of antibodies from a naive human or animal or population thereof and/or an antibody or population of antibodies from a dosed human or animal, and modifying the epitope to reduce the immune response to the polypeptide while still retaining a substantial therapeutic activity. The modified polypeptides are useful therapeutically.
The invention also includes methods and compositions useful to make modified polypeptides. The methods include a method of making a modified polypeptide recombinantly by modifying a nucleic acid sequence to encode a modified polypeptide, wherein the modified polypeptide has at least one change to an immunodominant epitope to reduce the immune response to the polypeptide while still retaining a substantial therapeutic activity. Compositions include modified nucleic acids and host cells comprising the modified nucleic acids.